Function of SLEEPLESS in Sleep Regulation Summary Sleep serves essential biological functions, and is conserved from flies to humans. The overall goal of the proposed research is to elucidate the molecular genetic basis of sleep regulation. Through an unbiased forward-genetic screen for short-sleeping mutant flies, we identified a novel gene, sleepless (sss), which is required for normal sleep as well as recovery sleep after deprivation. sss encodes a small, brain-enriched protein expressed on the cell surface. Expression of the Shaker (Sh) potassium channel, previously shown to be important for sleep regulation, is markedly reduced in sss mutants. Several key questions need to be addressed to understand how SSS regulate sleep. Does Sh mediate the effect of sss on sleep? Does SSS act directly on Sh by forming a complex or does it act indirectly via a signaling pathway? Does SSS act on Sh cell-autonomously or non-cell-autonomously? Do circadian and homeostatic processes affect SSS and Sh expression? To address these questions, we propose the following specific aims: (1) Test whether SSS promotes sleep by acting on Sh and whether they physically interact. To confirm a potential genetic interaction between sss and Sh, we will look for a synergistic effect on sleep of reducing both sss and Sh activity. We will also examine whether overexpression of Sh can rescue the short-sleeping phenotype of sss mutants. In addition, we will test whether SSS and Sh physically interact. (2) Identify the locus of action of sss. Identification of sss, which has profound effects on both baseline and rebound sleep, allows us an opportunity to explore the cellular substrates of homeostatic sleep regulation. We will examine the expression pattern of the SSS protein, and carry out rescue experiments using a collection of tissue-specific drivers and an sss transgene. Cellular substrates of sleep under normal and deprived conditions will be compared. (3) Determine if SSS acts on Sh cell-autonomously. To understand how SSS acts on Sh to regulate sleep, it is important to determine the locus of action of Sh as well as that of sss. We will determine the Sh expression pattern and tissue requirement using tissue-specific rescue and targeted RNAi knockdown experiments. In addition, we will test whether SSS can be secreted and whether secreted SSS can rescue the sleep phenotype of sss mutants. PUBLIC HEALTH RELEVANCE: Lack of good-quality sleep is a common problem that can lead to accidents and reduced productivity, and there is growing awareness among healthcare professionals and the public that sleep is a critical health issue. Elucidation of molecular mechanisms underlying sleep regulation may provide us with important clues as to how and why we sleep, and may ultimately lead to novel treatments for sleep disorders.